Summary: [unreadable] We have been examining new genetic polymorphisms in a drug metabolizing enzyme, CYP2C9, and the effects of polymorphisms on the dose of warfarin required to control cardiovascular (thromboembolytic) disease in patients treated with warfarin(Limdi 2007a). A lower dose of warfarin was also required in individuals with aberrant CYP2C9 alleles. We then examined the effects of these polymorphisms of CYP2C9 on serious and lifethreatening side effects (serious hemorrhage and death) in a prospective clinical study in patients. We have developed new rapid pyrosequencing genotyping tests for known or suspected defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) which occur in Caucasians and African Americans. These were used in a translational study: a large prospective clinical study of 500 Caucasian and minority African American patients on warfarin therapy to prevent thrombolytic disease over a 2 year period and the effect of polymorphisms on dose required and adverse life-threatening hemorragic events assessed. The presence of variant alleles of CYP2C9 conferred a 5-fold increase in risk for major hemorrhage before stabilization of dose, and a 2-fold risk even after stabilization of dose (Limdi et al, 2007b). We have also studied the ability of retinoid-related orphan receptors (ROR) on transcriptional upregulation of the human CYP2Cs in liver. Three genes, ROR alpha, beta, and gamma, are differentially expressed in liver, kidney, lung, muscle, brown fat, thymus, and brain and are involved in the regulation of many physiological processes such as immune function, brain development, circadian rhythm, and lipid metabolism. ROR and alpha1, 2, and gamma1 are expressed in liver. We examined whether RORs activate the promoter regions of the human CYP2C drug metabolizing enzymes in HepG2 cell (liver cell line) and a human colon carcinoma cell line, Caco 2 cells and whether they upregulate the mRNAs for the CYP2Cs. Overexpression of ROR and ROR in HepG2 cells increased luciferase activity of a 3kb CYP2C8 promoter construct, but not that of CYP2C9 or CYP2C19 promoter constructs. CYP2C8 is important in the metabolism of clinical drugs such as paclitaxel (breast cancer), certain antidiabetic drugs for type II diabetes, and antimalarial drugs as well as metabolizing arachidonic acid to EETs which are important in cardiovascular regulation. We identified the regulatory sites for ROR to one essential site in the CYP2C8 promoter using mutational assays, showed binding of RORs in gel-shift assays and binding to chromatin was shown in HepG2 cells using Chromatin immunoprecipitation (ChIP) assays. Overexpression of RORs using adenoviral constructs increased CYP2C8 mRNA in human primary hepatocytes as well as HepG2 cells. siRNA to ROR alpha and gamma decreased CYP2C8 mRNA expression in HepG2 cells. These data support a role of RORs in upregulation of CYP2C8 expression in liver and possibly extrahepatic tissues in response to stress, hypoxia and diurnal rhythm. Exposure of patients or human liver cells to clinical drugs transcriptionally upregulates CYP2C8, 2C9, and 2C19 enzymes and can produce tolerance to drugs or drug-drug interactions complicating patient therapy. In liver and intestine the CYP2Cs can be increased >2-8 fold by prior administration of drugs, producing higher metabolism in exposed individuals leading to tolerance. We have shown that the promoter regions of the human CYP2C genes are regulated by elements which bind the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and liver-enriched receptors such as HNF4 alpha. New data shows that HNF4 sites in the promoter enhance inducibility by CAR or PXR, and HNF4 and CAR act synergistically to increase CYP2C9 in liver cell lines. We theorize that various coactivators may be involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter. Results of yeast two hybrid screens identified NCOA6 as a new HNF4 interacting protein as well known coactivators such as PGC-1. We overexpressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4 and GST-CAR. CAR was identified as part of a complex with GST-HNF4 complex in nuclear extracts of HepG2 cells by Western blotting and by mass spectrometric analysis. Cofactors such as NCOA6 and PGC-1 were also identified in the complex. When NCOA6 and PGC-1 were translated in vitro, they could also interacted with HNF4-GST and were identified by Western blotting showing direct interaction. Using promoter assays and mRNA measurement, the synergy between HNF4 and CAR in the presence of their respective drug ligands could be blocked by adenoviral constructs to siRNAs for NCOA6, the new HNF4 interacting protein. In ChIP assays antibodies to CAR pulled down both the CAR binding site and the HNF4 binding site. NCOA6 was pulled down at the HNF4 site > the CAR site. Adenoviral constructs containing siRNA to NCOA6 did not affect HNF4 binding to its site but blocked NCOA6 binding and binding of other coregulators such as PGC-1, CBP, and PMT (an O-methyltransferase which repairs ASN and ASP residues). Therefore NCOA6 appears to be an essential cofactor that helps form bridge between CAR and HNF4 (the bridge may contain other coregulators as mentioned). Ongoing studies in primary human hepatocytes have shown that liver-specific HNF4 and HNF4 sites in the promoter of the CYP2Cs are required for maximum basal expression of the CYP enzymes and for PXR mediated induction of CYP2